Relevant to the present application is the inventors' own earlier international patent application WO 97/05265 (O'Hare and Elliott) (published after the priority date claimed for this application), which relates to VP22 protein and its properties and uses. Similarly the inventors' paper (Elliott and O'Hare (1997), in Cell, vol 88 pp 223-233 (1997), relates to intercellular trafficking and protein delivery by a herpesvirus structural protein. Both these documents are hereby incorporated in their entirety by reference and made an integral part of this disclosure.
The inventors have shown that the HSV-1 virion protein VP22 possesses an unusual intercellular trafficking mechanism, an effect particularly described in specification WO 97/05265. VP22 is a 38kDa protein which in primary-expressing transfected mammalian cells is located predominantly in the cytoplasm where it associates with cellular microtubules (see accompanying drawing, FIG. 1b). However a remarkable property of VP22 is its ability to spread throughout a monolayer of non-expressing cells. VP22 is transported from the cytosplasm of an expressing cell into neighbouring cells where it accumulates in the nucleus (FIG. 1b). The mechanism of this transport is still incompletely understood, but has been shown to be via a golgi-independent pathway and may utilise the acting cytoskeleton. HIV-1 Tat (Ensoli et al., 1993, Fawell et. al., 1994) and a small number of other non-viral proteins (Jackson et al., 1992) have been attributed with intercellular trafficking properties, but none appears to demonstrate this phenomenon as strikingly as VP22. A further important property of VP22 is that when applied exogenuously to the medium of an untransfected cell monolayer, it can be taken up by those untransfected cells where it accumulates in the cell nucleus.
The prior art generally includes a variety of antigens, immunomodulating proteins, proteins that are conditionally cytotoxic or lethal upon administration (to a cell containing them) of a corresponding drug or activator compound, proteins for cell cycle control, and other therapeutic and diagnostic proteins, especially in the forms of protein and polynucleotide sequences enabling genetic manipulation by standard techniques. References to some examples of these materials are given below.
For example, among cell cycle control proteins, protein p53 is known as a tumour suppressor, p53 is a 53kDa nuclear phosphoproprotein (FIG. 1c). Wild-type and mutant p53 proteins have been expressed by means of recombinant vaccinia viruses, (Ronen et al., Nucleic Acids Research, 20:3435-3441, 1992). p53 functions to regulate cell cycle progression and under conditions of DNA damage through a complex signal transduction mechanism can induce cell cycle arrest or apoptosis (Levine 1997). Failure to synthesize p53, or more commonly synthesis of a mutated form of the protein can result in uncontrolled cell proliferation and tumour formation. It has been shown by several groups that exogenous addition of functional wild type p53 can promote cell cycle arrest and/or apoptosis resulting in tumour regression with examples including cervical carcinomas (Hamada et al., 1996) and breast cancer xenografts (Nielsen et al., 1997). A number of p53 delivery systems have been utilised in vivo and in vitro such as intravenous injection of a p53:liposome complex (Kumar et al., 1997), direct transfection (Zheng et al., 1996) and adenoviral mediated transfer (Hamada et al., 1996, Sandig et al., 1997) but delivery of functional protein into a sufficiently high percentage of surviving cells remains a difficulty.
Also known from U.S. Pat. No. 5,484,710 (La Jolla: J C Reed et al) are regulatory elements linked to genes involved in cell death, as regulated by p53 tumour suppressor protein, and further proteins and their analogues for cell cycle control.
It remains desirable to provide particular further cell-delivery constructs for useful proteins.